Detection of Aggregation-Prone Behavior in Mutant P53 V157F Breast Cancer Cells Using Multipoint Thioflavin T Fluorescence

This study examines the aggregation propensity of mutant p53 V157F in breast cancer cells and evaluates multi-point plate reading for detection. Current experimental challenges include accurately detecting p53 aggregation, quantifying amyloid like structures, and ensuring consistent measurement across cell-based assay surfaces. To begin, take 10 microliters of trypan blue in a micro centrifuge tube.

Add 10 microliters of resuspended cells in DPBS to the same tube and mix the contents thoroughly to ensure homogeneity. Load 10 microliters of the prepared mixture onto a counter slide to count the live cells using an automated cell counter. Based on the viability count, seed 30, 000 viable cells per well into a 96 well culture plate.

Designate one well as a non-stained control and designate the remaining three wells for staining. Weigh 0.02 grams of Thioflavin T or THT powder using a balance. Add the powder to five milliliters of deionized water and mix thoroughly until the powder is completely dissolved.

Then add the staining components to one milliliter of DPBS. Add THT and Hoechst stock solutions to the tube and mix gently to ensure homogeneity. Now remove the culture medium from each well of the 96 well culture plate.

Add 100 microliters of the prepared THT and Hoechst staining buffer to each staining well followed by 100 microliters of DPBS to the non-stained control well. Place the culture plate in a dark place at room temperature for 30 minutes. Carefully aspirate or discard the staining solution from each well without disturbing the cell monolayer.

Then add 100 microliters of DPBS to each well and leave it for 30 seconds. Then discard the DPB. After repeating the washing step, add 100 microliters of fresh DPBS to each well.

Remove the cap from the 96 well culture plate before reading the fluorescent signal. Place the 96 well culture plate into the microplate reader. Measure THT fluorescence with excitation at 450 nanometers and a mission at 490 nanometers and measure Hoechst 33342 fluorescence with excitation at 360 nanometers and a mission at 460 nanometers.

Select the desired read area and then set the reader parameters, including shaking for five seconds before reading top redirection and an integration time of 140 milliseconds. After acquiring the fluorescence data, perform calculations to obtain the protein aggregation values. Finally normalize the THT fluorescence intensity by setting the ratio in MCF7 cells as one to serve as the baseline control Measurements were performed using both a single point and a four point method across stained and non-stained wells in MCF7 and Hs578T cells.

Hs578T breast cancer cells showed a 3.2 fold increase in thioflavin T fluorescence intensity compared to MCF7 cells using the single point reading method. When using the four point average reading method, Hs578T cells exhibited a 3.86 fold increase in thioflavin T fluorescence compared to MCF7 cells. Thioflavin T fluorescence in Hs578T cells was consistently elevated at all four measurement points compared to MCF7 cells.

This protocol enables accurate protein aggregation detection in cellular contexts using thioflavin T staining, combined with reliable multi-point fluorescence plate reading methods.